Not applicable.
The present invention relates to a transgenic non-human animal lacking native presenilin 1 (PS1) protein and a transgenic non-human animal expressing either the wild-type human PS1 or human PS1 containing a Familial Alzheimer""s Disease (FAD) mutation on native PS1 null background. The transgenic animal can be used in the study of the in vivo functions of PS1 and the effect of FAD mutation in PS1 function both during embryonic development and during aging. The transgenic animal can also be used in the identification of compounds that modulate the expression or activity of PS1.
Presenilin 1 (PS1) is a protein expressed in the central nervous system as well as other tissues of animals from early embryonic development through adult life. Endoproteolytically processed in vivo, (G. Thinakaran et al., Neuron 17:181 (1996)), PS1 is an eight transmembrane protein homologous to sel-12, a C. elegans protein that facilitates signaling mediated by the Notch/in-12 family of receptors (D. Levitan and I. Greenwald, Nature, 377:351 (1995), S. Artavanis-Tsakonas et al., Science 268:225 (1995)). In nematodes, particular egg-laying defects associated with loss of sel-12 function are rescued by PS1 (D. Levitan et al., Proc. Natl. Acad. Sci., USA 93:14940 (1996)), indicating that for some functions, the homologous proteins might be functionally interchangeable.
PS1 has been linked in Alzheimer""s Disease (AD), a neurological disorder that disproportionately affects the population over 65 years of age. Mutations in PS1 contribute to approximately 25% of early-onset familial Alzheimer""s Disease. Incidence of the disease increases from less than 1% at age 60-65, to 5% at age 75, to as high as 47% at age 85. As a result, 60% to 80% of all cases of dementia in persons over age 65 are caused by AD. Afflicted individuals exhibit impaired cognitive function and memory.
Distinguishing features of AD include the presence of senile plaques as well as, neurofibrillary tangles and extensive neuronal loss in the neocortex, hippocampus and associated structures. The senile plaques are extracellular deposits of heterogeneous substances of which the major component is a 39-43 amino acid peptide referred to as xcex2-amyloid peptide or Axcex2(Glenner and Wong,Biochem. Biophys. Res. Commun. 120:885-890 (1984). The 4 kDa Axcex2 peptide is derived by proteolytic cleavage of a larger xcex2-amyloid precursor protein (APP). The plaques are surrounded by a halo of dystrophic neurites, glia and astrocytes. xcex2-amyloid deposits are also present in neocortex blood vessel walls. Other components of the plaques include ubiquitin, amyloid P, Apo E, interleukin-1, and a-i-antichymotrypsin. Although the complete etiology of AD has not yet been determined, much is now known, including genetic, immunological and environmental factors implicated in the development of AD.
Genetic data is seen from the study of familial AD. Although the majority of AD cases appear sporadic, about 10% of cases are early onset familial AD (FAD). Genetic analysis of FAD families has established that the disorder is associated with autosomal dominant inheritance of mutations in specific genes including the xcex2-amyloid precursor protein (APP) located on chromosome 21, the PS1 gene on chromosome 14 and a homolog of PS1, presenilin 2 (PS2), located on chromosome 1. About 25% of the early-onset FAD cases are linked to mutations in PS1. Biochemical studies have shown that mutations in all three genes lead to an increased production of either the total Axcex2 or Axcex242(43), which is believed to be more amylodogenic (Scheuner et al., Nature Medicine 2:864-870 (1996); Selkoe, Science, 275:630-631 (1997)). To date, 42 different missense mutations and one in-frame splice site mutation were described (Cruts et al, Hum. Mol. Genet. 5:1449-1455 (1996)). However, neither all of the physiological roles of PS1, nor its mechanism in AD pathogenesis, are fully understood at this time.
Transgenic non-human animal technology offers a model system to address the effects of genes associated with AD. Addition of a gene construct directing the expression of human PS1 or its components to key regions in the central nervous system provide a means to study the expression and activity of PS1 and modulators of PS1. Previous attempts to express either the wild-type human PS1 protein or human PS1 containing various FAD mutations under either the PDGF promoter or the PrP promoter have been reported (Duff et al., Nature 383:710-713 (1996); Borchelt et al., Neuron 17:1005-1013 (1996)). In each case, the PS1 transgenes were reportedly expressed on the wild-type murine PS1 background. Thus, a mixture of murine and human PS1 proteins were produced in the resulting transgenic mice.
A relative increase of Axcex242(43) level has been observed in plasma and fibroblast cell cultures derived from PS1 FAD subjects and in transgenic mice expressing human PS1 FAD proteins (Scheuner et al, 1996; Duff et al., (1996); Borchelt et al, (1996); Lemere et al., (1996); Citron et al., Nature Medicine 3:67-72 (1997)). That result suggests that the pathogenic consequence of PS1 mutations might be through APP processing and Axcex242(43) production.
Accordingly, it is an aspect of this invention to provide a non-human transgenic animals which is heterozygous for a functional PS1 gene native to that animal. As used herein, functional is used to describe a gene or protein that, when present in a cell or in vitro system, performs normally as if in a native or unaltered condition or environment. The animal of this aspect of the invention is useful for the study of the tissue and temporal specific expression or activity of PS1 in an animal having only one functional copy of the gene. The animal is also useful for studying the ability of a variety of compounds to act as modulators of PS1 activity or expression in vivo or, by providing cells for culture, in vitro. As used herein, a modulator is a compound that causes a change in the expression or activity of PS1, or causes a change in the effect of the interaction of PS1 with its ligand(s), or other protein(s). In an embodiment of this aspect, the animal is used in a method for the preparation of a further animal which lacks a functional native PS1 gene. In another embodiment, the animal of this aspect is used in a method to prepare an animal which expresses a non-native PS1 gene in the absence of the expression of a native PS1 gene. In particular embodiments the non-human animal is a mouse. In further embodiments the non-native PS1 is a wild-type human PS1, an A246E mutant human PS1 gene or any other mutant human PS1 gene.
In reference to the transgenic animals of this invention, we refer to transgenes and genes. As used herein, a transgene is a genetic construct including a gene. The transgene is integrated into one or more chromosomes in the cells in an animal by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal. A gene is a nucleotide sequence that encodes a protein. The gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art.
Another aspect of the invention is a non-human animal embryo deficient for native PS1 expression. This embryo is useful in studying the effects of the lack of presenilin 1 on the developing animal. In particular embodiments the animal is a mouse. The animal embryo is also useful as a source of cells lacking a functional native PS1 gene. The cells are useful in in vitro culture studies in the absence of PS1.
An aspect of this invention is a method to obtain an animal in which the cells lack a functional gene PS1 native to the animal. The method includes providing a gene for an altered form of the PS1 gene native to the animal in the form of a transgene and targeting the transgene into a chromosome of the animal at the place of the native PS1 gene. The transgene can be introduced into the embryonic stem cells by a variety of methods known in the art, including electroporation, microinjection, and lipofection. Cells carrying the transgene can then be injected into blastocysts which are then implanted into pseudopregnant animals. In alternate embodiments, the transgene-targeted embryonic stem cells can be coincubated with fertilized eggs or morulae followed by implantation into females. After gestation, the animals obtained are chimeric founder transgenic animals. The founder animals can be used in further embodiments to cross with wild-type animals to produce F1 animals heterozygous for the altered PS1 gene. In further embodiments, these heterozygous animals can be interbred to obtain the non-viable transgenic embryos whose somatic and germ cells are homozygous for the altered PS1 gene and thereby lack a functional PS1 gene. In other embodiments, the heterozygous animals can be used to produce cells lines. In preferred embodiments, the animals are mice.
A further aspect of the present invention is a transgenic nonhuman animal which expresses a non-native PS1 on a native PS1 null background. In particular embodiments, the null background is generated by producing an animal with an altered native PS1 gene that is non-functional, i.e. a knockout. The animal can be heterozygous (i.e., having a different allelic representation of a gene on each of a pair of chromosomes of a diploid genome) or homozygous (i.e., having the same representation of a gene on each of a pair of chromosomes of a diploid genome) for the altered PS1 gene and can be hemizygous (i.e., having a gene represented on only one of a pair of chromosomes of a diploid genome) or homozygous for the non-native PS1 gene. In preferred embodiments, the animal is a mouse. In particular embodiments the non-native PS1 gene can be a wild-type or mutant allele including those mutant alleles associated with a disease. In further embodiments, the non-native PS1 is a human PS1. In particular embodiments the PS1 gene is a human wild-type PS1 allele, the human A246E mutant allele or another mutant human PS1. In further embodiments the non-native PS1 gene is operably linked to a promoter. As used herein, operably linked is used to denote a functional connection between two elements whose orientation relevant to one another can vary. In this particular case, it is understood in the art that a promoter can be operably linked to the coding sequence of a gene to direct the expression of the coding sequence while placed at various distances from the coding sequence in a genetic construct. In a preferred embodiment, the promoter is a neuronal specific human Thy-1 promoter. Further embodiments are cells derived from animals of this aspect of the invention.
An aspect of this invention is a method of producing transgenic animals having a transgene including a non-native PS1 gene on a native PS1 null background. The method includes providing transgenic animals of this invention whose cells are heterozygous for a native gene encoding a functional PS1 protein and an altered native PS1 gene. These animals are crossed with transgenic animals of this invention that are hemizygous for a transgene including a non-native PS1 gene to obtain animals that are both heterozygous for an altered native PS1 gene and hemizygous for a non-native PS1 gene. The latter animals are interbred to obtain animals that are homozygous or hemizygous for the non-native PS1 and are homozygous for the altered native PS1 gene. In particular embodiments, cell lines are produced from any of the animals produced in the steps of the method.
The transgenic animals and cells of this invention are useful in the determination of the in vivo function of a non-native PS1 in the central nervous system and in other tissues of an animal. The animals are also useful in studying the tissue and temporal specific expression patterns of a non-native PS1 throughout the animals. The animals are also useful in determining the ability for various forms of wild-type and mutant alleles of a non-native PS1 to rescue the native PS1 null deficiency. The animals are also useful for identifying and studying the ability of a variety of compounds to act as modulators of the expression or activity of a non-native PS1 in vivo, or by providing cells for culture, for in vitro studies.
An aspect of this invention is an assay to detect the effect of a compound on Axcex2 production. In a preferred embodiment, the assay is performed by providing an animal of the present invention, exposing the animal to the compound, and measuring Axcex2 production in the animal. The measurement can be compared to a measurement of Axcex2 production in a genetically similar or identical animal that is not exposed to the compound. In another preferred embodiment, the assay is conducted as above except that one uses populations of cells derived from an animal of this invention in place of the animals themselves. In conducting the assays of this invention, the animals or cells are exposed to compounds at various dosages over various periods of time. The effect of a compound on Axcex2 production can be measured by a variety of methods known to those of skill in the art including sandwich ELISA, western blot analysis and mass spectrometry. The term xe2x80x9cexposurexe2x80x9d is used in it""s ordinary sense and includes exposing cells to a compound by placing the compound in their environment, e.g., in the medium, and includes exposing animals to a compound by injection, inhalation, consumption, external or internal application, supository and other means known in the art.